The stimulatory G protein (GS) mediates the activation of hormone- responsive adenylyl cyclase. Gs consists of an alpha subunit (Gs-alpha) which has an intrinsic GTPase activity, and beta and gamma subunits that are tightly associated. It has been proposed that during activation of Gs, Gs-alpha binds to GTP, dissociates from beta-gamma, and interacts with adenylyl cyclase to stimulate the enzyme. We purified Gs from bovine brain, coupled a radiolabeled, heterobifunctional cross-linking agent to it, and reconstituted it into S49 cyc membranes which lack Gs- alpha. This protocol will be useful for determining which subunits of Gs interact with adenyl cyclase under different conditions. We also have expressed a recombinant Gs-alpha(rGs-alpha)as a fusion protein in bacteria in order to simplify the preparation of large quantities of this protein. The fusion protein was purified by affinity chromatography and cleaved by proteolysis. The resultant rGs-alpha has GTPase activity but differs from Gs-alpha by having six additional amino acids on its N-terminal. It was incorporated into S49 cyc membranes but was unable to reconstitute hormone-responsive adenylyl cyclase activity. We are investigating why rGs-alpha is inactive. The carbohydrate moieties of the glycopeptide hormone human chorionic gonadotropin (hCG) have N-acetyl-neuraminic acid (NeuAc) residues that are required for full biological activity. We use hCG stimulation of adenylyl cyclase in MLTC-1 cells as a model to study the role of sialic acid in the biological activity of hCG. Removing NeuAc produced asialo-hCG which had only 50% of the biological activity of hCG. NeuAc with an azido group on carbon atom nine was synthesized and enzymatically coupled to asialo-hCG to produce 9-azido, 9-deoxysialyl hCG. The modified hormone was radiolabeled for use as a photoaffinity probe to investigate the interaction between carbohydrate moieties of hCG and the gonadotropin receptor.